All aspects of cardiovascular function are regulated by receptors of the seven transmembrane receptor (7TMR) family. The largest of all the receptor families, it includes receptors for catecholamines, acetylcholine, angiotensin, adenosine and endothelins. A universal mechanism regulating these receptors is desensitization of heterotrimeric G-protein signaling. Classically, this is mediated by a two-step process in which activated receptors are phosphorylated by G-protein-coupled receptor kinases (GRKs) leading to the binding of a - arrestin ( arr) molecule, which sterically interdicts further activation of the G-protein. More recently it has become clear that arrs can also serve as multifunctional endocytic and signaling adaptors, which can also activate additional pathways involved in such physiological outcomes as chemotaxis and anti-apoptosis, thus regulating cardiovascular function in atherosclerosis, restenosis, and cardiac hypertrophy. Moreover, for several receptors, including the 1 and 2-adrenergic receptors (ARs) and angiotensin 1A receptor (AT1AR), amongst others, ligands can be found which disproportionately activate either G-protein or arr mediated signaling - i.e. biased ligands. Such compounds have potentially unique therapeutic properties. Accordingly, this proposal has three closely linked aims, which involve a focus on elucidating the molecular mechanisms by which 2arrs mediate signaling by 7TMRs. Our goals are: 1) to elucidate the full extent of arr mediated signaling networks downstream of cardiovascular 7TMRs such as the AT1AR and 2-AR using the approaches of global cellular phosphoproteomics and systems biology; 2) to develop arr-biased ligands for the 2-AR via high throughput and targeted screening; and 3)to elucidate the molecular and biophysical basis of such signaling in terms of ligand specific conformational alterations in arrs via analysis of receptor phosphorylation sites, development of conformationally specific antigen binding fragments of antibodies (Fabs) and crystallographic techniques. Our hypothesis is that the phosphorylation of distinct sites on receptors by different GRKs leads to structurally and functionally distinct activated conformations of the receptors and arrs which mediate distinct signaling outcomes. By understanding the functional signaling consequences and structural basis of arr-biased agonism we will lay the basis for the development of a novel class of therapeutic agents. PUBLIC HEALTH RELEVANCE: Relevance Some of the most important drugs used to treat cardiovascular disease such as blockers ( -adrenergic receptor antagonists) or ARBs (angiotensin receptor blockers) work through specific receptors on the outside of cells. Here we seek to understand, at a molecular level, a newly discovered mechanism by which these receptors signal to the inside of the cell, in order to lay the basis for an entirely novel class of therapeutic agents for the treatment of cardiovascular and other diseases.